Elution and identification of T cell epitopes from viable cells

Abstract

Methods are provided for eluting peptides that are bound to major histocompatibility complex ("MHC") molecules expressed on the cell surfaces of viable cells that have at least one MHC-peptide complex on the surfaces of the cells, the method comprising incubating the cells in the presence of peptide elution buffer, preferably comprising iso-osmotic, citrate-phosphate buffer at a pH of approximately 3.3, for between about 15 seconds and one minute. Using these methods a naturally processed melanoma peptide recognized by CD8+ cytotoxic T lymphocytes has been identified.

We claim: 
     
       1. A pharmaceutical composition, comprising: T cell epitopes consisting of peptides which have been presented by major histocompatibility complex ("MHC") molecules expressed on the cell surface of viable cells and which do not need further processing for subsequent presentation by MHC molecules to allow for T cell immune recognition, obtained by eluting said T cell epitopes from said cells, said method comprising the steps of: incubating said cells in the presence of peptide elution buffer such that said cells remain viable; and   recovering said T cell epitopes from said peptide elution buffer.     
     
     
       2. The pharmaceutical composition of claim 1, wherein said T cell epitopes are selected from the group consisting of fractions P1, P2 and P4 fractionated on an HPLC linear gradient of from 0% acetonitrile/99.92% water/0.08% trifluoroacetic acid to 35% acetonitrile/64.93% water/0.07% trifluoroacetic acid such that P1 elutes at an acetonitrile concentration of about 19-21.5% acetonitrile; P2 elutes at an acetonitrile concentration of about 21.5-23.5% acetonitrile; and P4 elutes at an acetonitrile concentration of about 25-25.5% acetonitrile. 
     
     
       3. The pharmaceutical composition of claim 1, wherein said T cell epitopes are selected from the group consisting of fractions P1-P6 fractionated on an HPLC linear gradient from 0% acetonitrile/99.92% water/0.08% trifluoraoacetic acid to 35% acetonitrile/64.93% water/0.07% trifluoroacetic acid such that P1 elutes at an acetonitrile concentration of about 19-21.5% acetonitrile; P2 elutes at an acetonitrile concentration of about 21.5-23.5% acetonitrile; P3 elutes at an acetonitrile concentration of about 23.5-24.5% acetonitrile; P4 elutes at an acetonitrile concentration of about 25-25.5% acetonitrile; P5 elutes at an acetonitrile concentration of about 25.5-26.5% acetonitrile; and P6 elutes at an acetonitrile concentration of about 26.5-28.5% acetonitrile. 
     
     
       4. The pharmaceutical composition of claim 3, wherein said T cell epitopes are selected from the group consisting of peaks 1, 2, and 3 obtained from HLA-A2+ melanoma cells wherein said peaks are shown in FIGS. 9A-9C. 
     
     
       5. The pharmaceutical composition of claim 4, wherein said melanoma cells are selected from the group consisting of Mel 9742 and Mel 624. 
     
     
       6. The pharmaceutical composition of claim 5 wherein said T cell epitope consists of a synthetic peptide having the sequence identified for the peptide p939 (SEQ ID NO: 38). 
     
     
       7. A T cell epitome consisting of T cell epitope p939 (SEQ ID NO: 38), wherein said T cell epitope is presented by HLA-2 molecules and recognized by cytotoxic T lymphocytes.